Tissue specific stem cells (TSSCs) are rare precursor cells in adult tissues that divide to produce progeny cells that mature into functional tissue cells. In this way, adult stem cells function to replenish mature tissue cells that have expired or to repair tissues and organs that are diseased or damaged. These natural restorative properties make adult stem cells attractive for development for use in gene therapy and cell replacement therapy.
To perform their tissue renewal functions, stem cells must produce differentiating cells while simultaneously maintaining their own undifferentiated, proliferative state. This essential adult stem cell property, called asymmetric cell kinetics (also known as asymmetric self-renewal), is also a major barrier to the detection, counting, expansion and isolation of these cells in culture or in a population of cells derived from a primary tissue. The reason for this difficulty is that in propagating TSSCs in culture and during proliferation, TSSCs are lost in the sea of differentiating cells that they produce. Methods that facilitated effective and accurate estimation of the number of TSSCs in a population of proliferating cells would aid in research and development of gene therapy and cell replacement therapy that uses TSSCs.
Several methods are available for detecting and counting TSSCs. For example, Lee et al., (2003, Biotechnol. Bioeng. 2003, 83:760-71) describes a method for expanding and quantifying TSSCs ex vivo, based on their unique property of asymmetric self-renewal. When TSSCs undergo asymmetric self-renewal, their divisions produce another actively cycling TSSC and a non-stem sister cell. The non-stem sister cell undergoes differentiation, which in culture is often accompanied by cell cycle arrest. The asymmetry for cell cycle transit by the TSSC sister and the non-stem sister cell can be scored by pulse-labeling the paired cells with the thymidine analogue bromodeoxyuridine (BrdU). Since the stem cell sister continues through S-phase, it incorporates BrdU; but the non-stem sister arrests in G1 phase before S phase, and, therefore, does not incorporate BrdU. The asymmetric pattern of sister cell BrdU uptake is specific for the division of asymmetrically self-renewing TSSCs. Both symmetrically self-renewing TSSCs (which occur during body maturation and potentially during wound repair) and symmetrically cycling transiently amplifying, differentiating cells show symmetric patterns of BrdU uptake, with both sisters being BrdU(+). Therefore, this method is not applicable to symmetrically self-renewing TSSCs.
Incorporated BrdU is detected by conventional in situ immunofluorescence (ISIF) with fluorescently tagged BrdU-specific antibodies or by visualizing the quenched fluorescence of DNA dyes like Hoechst. This “sister pair analysis” (SPA or SP) requires that cells are evaluated at cell densities that are sufficiently low to allow verification of sister-sister relationships. Therefore, SPA is not applicable at the high cell densities needed to investigate TSSCs in heterogeneous tissue cell preparations. Moreover, the use of cytochalasin D to trap sister nuclei in the same cytoplasm to verify their sister-sister relationship at high cell densities, as used in the U.S. Patent Application No. 2005/0074874, is not applicable for BrdU-SPA because asymmetric BrdU uptake is lost in cytochalasin D-arrested binucleates.
The U.S. Patent Application No. 2005/0074874 describes the use of immortal DNA strand co-segregation (IDSC) as a specific indicator of TSSCs. It scores the ability of TSSCs to continuously co-segregate chromosomes that bear their oldest DNA strands. This method does make use of cytochalasin D to verify sister-sister relationships even at high cell densities. In this case, BrdU is introduced into TSSC nuclear DNA according to either of two strategies before cells are treated with cytochalasin D. In the “label-exclusion” strategy, it is incorporated into all DNA strands except the immortal DNA strands as TSSCs undergo continuous asymmetric self-renewal. In the “label-retention” strategy, it is first incorporated into “pre-immortal DNA strands” when TSSCs either undergo physiological symmetric self-renewal or are induced to undergo symmetric self-renewal (during which chromosome segregation is random). Thereafter, BrdU-labeled cells are evaluated after a switch to asymmetric self-renewal for a BrdU-free chase. Thereafter, treatment of cells in either strategy with cytochalasin D yields binucleated cells with the BrdU-asymmetry signature of TSSCs. However, this method is not applicable in situations where TSSCs fail to exhibit IDSC.
Merok et al., 2002 (Cancer Res. 2002, 62:6791-5) and Rambhatla et al., 2005 (Cancer Res. 2005, 65:3155-61) both described similar methods to that of the U.S. Application No. 2005/0074874. In the former case, the TSSC nucleus has half the BrdU content of the non-stem sister nucleus; and in the latter case, the TSSC nucleus is the only nucleus that contains BrdU. A shortcoming of this “BrdU-Cyto D” method and the BrdU-SPA method is that both require BrdU incorporation, which can perturb normal cellular physiology and induce technical artifacts.
Other methods have been developed to detect and isolate stem cells using antibodies or peptides that specifically bind to cell surface marker proteins (Uchida N., et al., Proc. Natl. Acad. Sci. USA 2000, 97:14720-14725; Kiel M J, et al., Cell 2005, 121:1109-1121; Lawson, et al., Proc Natl Acad Sci USA 2007, 104:181-186; Morita T, et al. Biotechnol. Prog. 2006, 22:974-978) or based on transfection of plasmid with the promoter and reporter genes in the case of induced pluripotent stem cells (Kim J H, et al. Nature 2002, 418:50-56; Yoshizaki T, et al., Neurosci. Lett. 2004, 363:33-37). The latter transgenic detection method suffers from low sensitivity; since it can only detect cells are that effectively transfected. Won Jong Rhee and Gang Bao 2009 (BMC Biotechnology 2009, 9:30) described a method for detecting and isolating of stem cells by targeting both an intracellular marker (mRNA) and a cell surface marker (SSEA-1 protein). The Wnt target gene Lgr5 has been recently identified as a novel stem cell marker of the intestinal and colonic epithelia and the hair follicle. However, it is unclear whether Lgr5 is expressed specifically by tissue stem cells or the more abundant immediate committed progenitor cells that they produce (Snippert et al., Cell 2010, 143:134-44). This uncertainty highlights a common limitation of all described methods based simply on “TSSC marker” expression, whether endogenous or transgenic. The markers are not exclusive to TSSCs, but are also expressed by their immediate cell products, which are committed to differentiation.